大鼠胚胎神经干细胞的分离培养及鉴定研究

Study of Cell Detachment,Cultivation and Identification of the Rat Embryo Neural Stem Cells in Vitro

目的探讨大鼠胚胎神经干细胞（NSCs）的分离、培养、分化及鉴定的条件与方法。方法分离孕14～16dSD大鼠胚胎脑皮质及皮质下区域NSCs，无血清培养基（DMEM／F12，含bFGF、EGF、B27等）条件下培养，通过有限稀释法克隆、传代，含血清培养基诱导分化，免疫组织化学法鉴定NSCs及诱导分化后的细胞。结果体外培养的NSCs能稳定增殖成神经干细胞球并传代（nestin染色阳性），经诱导可分化为神经元细胞（NSE染色阳性）、星形胶质细胞（GFAP染色阳性）和少突胶质细胞（CNP染色阳性）。结论采用无血清培养基中加入特定生长因子的培养技术，可在体外大量培养出稳定增殖并具有多向分化潜能的大鼠胚胎NSCs，为进一步研究NSCs的生物特性及应用奠定了基础。

Objective To explore the conditions and methods of detachment, cultivation, differentiation and identification of the rat embryo neural stem cells （NSCs） in vitro. Methods NSCs were derived from the cortex and suhcortex tissue of 14 to 16 days aged rat embryo and cultured in the serum-free medium which comprised epidermat growth factor （EGF）, basic fibroblast growth factor （bFGF） and B27. The cells were cloned and passaged through limiting dilution assay and differentiatiated in 10% fetal calf serum. The rat neural stem cells and the differentiated neural stem cells were identified respectively by immunofluorescenee including nestin, neuron specific enolase （NSE）,gilial fihrillary acidic protein （GFAP） and cyclieneucleotide phosphodiesterase （CNP）. Results The NSCs cultured in vitro could proliferate steadly and passage,which had been proved by positive nestin staining, and could be induced to differentiate into neurons （NSE positive）,astrocytes （GFAP positive） and oligodendrocyte （CNP positive）. Conclusion The rat NSCs have potency of multiple differentiation and can be successfully cultured in large scale in vitro when cultured in the serum-free medium with addition of growth factors, which provides the foundation for further research on biological characteristics and application of NSCs.